Abstract While a sterilizing cure of HIV-1 infection has only been reported in a single patient after a stem cell transplant with CCR5?32 homozygous cells, a spontaneous functional cure of HIV-1 occurs in 0.3-0.5% of all infected persons. These individuals, termed elite controllers (EC), maintain undetectable levels of HIV-1 replication in the absence of treatment, despite the repeated isolation of replication-competent virus from their serum. In this way, these individuals provide living evidence that immune-mediated control of HIV-1 infection is possible, and the identification of effective immune defense mechanisms that are active in these patients holds promise for inducing a functional cure of HIV-1 infection in a broader HIV-1 patient population. Previously, the analysis of such mechanisms has mostly focused on studying individual components of the immune system in an isolated fashion, however, it is now increasingly clear that effective immune defense programs in these patients are likely to involve complex networks of innate and adaptive immune responses and that integrative, iterative analysis steps will be required to mechanistically understand synergistic networks of immune defense in EC. Yet, such integrated programs of immune control can hardly be detected using traditional reductionist approaches that are biased towards specific pre-defined molecules or investigate one specific aspect of immune defense in an isolated fashion. Here, we will employ a multi-step research strategy to identify comprehensive, multi-system programs of immune defense in EC and explore their underlying functional mechanisms. In specific aim 1, we will use novel, high throughput technologies such as genome-wide SNP analysis and multiplexed mRNA, miRNA and protein expression analysis in sorted leukocellular subsets to identify specific genomic, transcriptional and proteomic characteristics uniquely associated with an elite controller phenotype. These assays will be performed in combination with multidimensional immunologic assays to identify functional signatures of innate and adaptive immune responses that are selectively observed in elite controllers (specific aim 2). Subsequently, a number of different biocomputational algorithms will be used to detect connectivity between various aspects of immune defense mechanisms, and identify holistic, integrative and unifying programs that link gene/protein expression, immune responses and clinical development of EC phenotype; these pathways will then be studied in detail using cutting-edge single-cell analysis approaches to identify and characterize the molecular circuits most suitable and promising for therapeutic manipulation (specific aim 3). By comprehensively exploring HIV-1 immune responses in EC in relationship to genetic variation and mRNA/miRNA/protein expression profiles and using this data to develop integrative models of HIV-1 immune defense, the proposed studies will generate unprecedented insights into effective mechanisms of HIV-1 immune control, and may lead to novel clinical strategies to induce a functional cure of HIV-1 in a broader patient population.